The present invention is especially pertinent to apparatus and methods for performing nanoparticle tracking analysis (“NTA”).
Nanoparticle tracking analysis is a relatively recently developed method for the direct and real-time visualisation and analysis of nanoparticles in liquids (see e.g. WO 03/093801). Based on a laser-illuminated microscopical technique, Brownian motion of nanoparticles is analysed in real-time by, for example, a charge-couple device (CCD) camera or the like, each individual particle being simultaneously but separately visualised and tracked by a dedicated particle tracking image-analysis programme. The ability of NTA to measure simultaneously particle size and particle scattering brightness allows heterogeneous particle mixtures to be resolved and, importantly, particle concentration to be estimated directly, the particle size distribution profile obtained by NTA being a direct number/frequency distribution.
NTA has become a term of art, recognised by those skilled in the relevant field. There are over 900 scientific papers and presentations referring to data collected using NTA. Further the term is used by, for example, ASTM International (formerly the American Society for Testing and Materials), the Environmental Protection Agency (EPA), the Food and Drug Administration (FDA) and the NIH.
The range of particle sizes that can be analysed by NTA depends on the particle type. The lower size limit is defined by the particle size and particle refractive index, given that sufficient light must be scattered by each particle for it to be detected and tracked as described above. For particles with very high refractive indices, such as colloidal gold, accurate determination of size can be achieved down to particles with a maximum dimension of about 10 nm. For lower refractive index particles, such as those of biological origin, the smallest detectable size might be in the range 25-50 nm Accordingly, NTA is limited by its ability to detect particles below a certain size.
With NTA, the presence and analysis of particles, each of which scatters sufficient light to be detected individually, can still be carried out even in the presence of ‘background’ material comprising, for instance, a population of very small particles (such as protein molecules, sub-10 nm inorganic material, polymer solutions, nano-emulsions, etc.) each of which is too small to detect individually but which is present in sufficiently high concentration to collectively form a background haze of scattered light. This background cannot be analysed by NTA, but particles visible as discrete light scattering entities embedded within this background may be analysed by NTA. Of course, the intensity of this background will determine the limit of sensitivity of NTA in terms of minimum detectable size. Further, NTA is able to identify, track and analyse suitably sized particles even when they are present in heterogeneous samples containing low numbers of larger particles.
NTA is further capable of detecting and analysing inherently fluorescent or fluorescently-labelled nanoparticles in the presence of a non-fluorescent background through use of appropriate fluorescence exciting optical sources and suitable fluorescence filters. NTA is further capable of measuring more than one fluorescence wavelength within a sample using multiple filters or a colour camera.
The sizing of individual nanoparticles by NTA is based on the analysis of Brownian motion exhibited by micron and sub-micron particles suspended in a liquid when illuminated by a suitable light source (e.g. laser) such that the light scattered by the particles is detected by a microscopical arrangement which causes some of the light scattered by the particles to be imaged by a video camera (typically CCD, electron multiplying CCD [EMCCD], scientific complementary metal oxide semiconductor [sCMOS] etc.).
The average distance moved by any given particle at known time intervals (e.g. the reciprocal of the frame rate of the camera, typically 30 frames per second) is related through the Stokes Einstein equation in which the diffusion coefficient can be extrapolated to particle hydrodynamic diameter, if the temperature and viscosity of the surrounding liquid are known.
The region of laser beam interrogated by the camera is a function of the size (in the x and y dimensions) of the image captured by the microscopical optical train onto which is fitted a suitable camera. For usual applications, a ×20 long working distance microscope objective is used. Because Brownian motion is effectively independent of particle mass, NTA (like the related technique of Dynamic Light Scattering) is considered an absolute technique, not requiring calibration. Because it interrogates particles individually within a suspension (though simultaneously), it is possible to generate high resolution particle size distribution profiles.
The field of view of the camera is typically about 100×80 microns and the depth of beam has previously been assumed to be approximately 10 μm.
However, the spatial dimensions (including “depth”) of the laser beam in which any given particle is visible to the camera (the ‘effective scattering volume’ or observation volume) is, especially given the frequently non-uniform intensity profile of the laser sources used, dependent on a variety of factors. These include the inherent sensitivity of the camera (adjustable by varying gain and shutter settings), the power and wavelength of the laser beam and, most importantly, is a strong function of the size and refractive index of the scattering particles.
The illuminating laser beams are not usually top-hat in profile (i.e. of uniform intensity throughout both the x and y dimensions) but are complex, ranging from a smooth Gaussian (or similar) profile to very complex profiles in which unpredictable spatial variations in cross-sectional intensity arise from the optical perturbations on launching the beam into the scattering cell through a glass wall at low (close to critical) angle.
Accordingly, while is it possible to dynamically determine with some confidence the particle size distribution of the particles successfully tracked by NTA, accurate estimation of the number of particles of any given size or size class present in the path of the laser beam is more problematical.
Put simply, smaller and/or lower refractive index particles are often only visible (to the camera) in the regions of the beam in which the incident intensity is highest (e.g. the centre of a Gaussian beam or brighter parts of, for example, a striated beam) and are not visible in lower intensity regions. In contrast, larger (or higher refractive index) particles can be seen at greater distances from the high intensity beam centre (or between high intensity striations) because they scatter more light. Thus for two such particle types the volume in which they are visible (the “effective observation volume”) will be different and consequently, even though they may be actually present in the same number concentration, NTA will detect different numbers of particles in the same system.
WO 2012/004320 discloses a method by which the effective observation volume can be determined by measuring the average track length of a particle moving under Brownian motion in a beam. With knowledge of the temperature and viscosity in which a particle of known size (or, more accurately, known diffusion coefficient) is moving, the length of time such a particle will be, on average, present and therefore scattering detectable amounts of light and thus trackable, will depend (all other things being equal) on the volume of the interrogation region. Larger effective ‘observation volumes’ result in longer track-lengths for any given sized particle.
In other words, the larger the beam (or more effective the particle is as a scatterer), the longer will be a particle's visible lifetime in the beam. If the size of a particle is known (e.g. because using a calibration particle) and the temperature and viscosity of the solvent is known, it is possible to calculate the volume of the beam from determining the track-length distribution of a monodisperse population of particles of a known size. Using this ‘absolute’ method by which the scattering volume can be spatially calculated, it is possible to determine the number of particles seen in a volume calculated, allowing generation of an absolute value for number concentration of the sample. However the limitations of this technique are that it assumes a continuous and uniform observation volume, and requires an accurate measurement of track length which could be significantly affected at high particle concentrations, with a high image noise or with a flowing sample.
Another technique for calibrating NTA apparatus is disclosed by Gardiner et al (“Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis” Journal of Extracellular Vesicles 2013, 2 1-11). This involves the use of a calibration sample of a particular nanoparticle population of known concentration, having similar characteristics (in terms of particle size and refractive index) to a particle population to be analysed.
However, the method is of limited practical usefulness, requiring a calibrant particle with appropriate properties (which may not always be available) and, in any event, the apparatus requires re-calibration each time if it is to be used to analyse a population of particles with different properties.
The present invention aims to reduce or overcome one or more of the problems associated with the prior art.